Chaperones are a group of proteins that assist in the folding and refolding of other intracellular proteins. There are many kinds of molecular chaperones HSP100, HSP90, HSP70, Chaperonin (HSP60), DNAJ (HSP40), etc.
One particular family of Chaperones, the Chaperonins, is conserved in all organisms, eukaryotes, archaebacteria and eubacteria alike. The most well studied protein in this family is the eubacteria protein GroEL which has served as a model system for determining the mode of action of the chaperoning.
GroEL exists as a homopolymeric structure in the form of a double ring or toroid structure composed of 7 identical subunits per toroid. The double toroid binds to denatured or partially unfolded proteins and during repeated rounds of ATP hydrolysis achieves the correct folding of the bound protein. The ATPase active site of the individual subunits represents the most highly conserved region of the Chaperonin family of molecules and clearly this function is critical to the activity of Chaperonins from all species. When examining the primary sequence similarity across the Chaperonin family it is apparent that whilst the ATPase motif is highly conserved (Kim et al. Trends Biochem Sci., 1994; Kubota at el, Gene 154, 231–236, 1995a) outside this region there is only moderate or weak homology between the prokaryotic or endosymbiotically derived type I Chaperonins, GroEL, HSP60 and RBP and the type II Chaperonins of archaebacterium and eukaryotes namely TF55, Thermosomes and CCT (TCP1).
The generally accepted role for GroEL is that it binds to exposed hydrophobic regions of polypeptides that are normally buried within the cores of soluble proteins. By binding to the exposed hydrophobic regions the GroEL prevents aggregation between the unfolded protein monomers themselves or other intracellular molecules. Following substrate binding to GroEL, cycles of ATP hydrolysis drive the progression of the bound substrate towards a folded or near folded state which is then released from the folding complex. GroEL appears to be able to bind to many denatured proteins by means of interaction with hydrophobic pockets or clefts on the surface of the GroEL, indeed GroEL is able to bind to some 50% of denatured cytosolic proteins (Viitanen et al, Protein. Sci. 1, 363–369, 1992), which suggests a broad specificity for hydrophobic regions in substrate proteins. GroEL mediated folding and release of many substrates is facilitated by the ring co-chaperonin GroES which caps the active cis side of the folding complex (Weissman et al, Cell 84, 481–490, 1996).
By analysis the Type II Chaperonin from eukaryotes, CCT, appears to be an wholly different molecule to GroEL for a number of obvious structural and less obvious mechanistic reasons. CCT is a heteropolymeric complex comprised of eight different subunits in each of two rings which exist as a double toroid structure, the eight subunits being encoded by eight different genes. CCT also appears to bind a far more restricted spectrum of partially folded substrates than GroEL. CCT appears to primarily interact with proteins of the cytoskeleton, namely actin and tubulin, and indeed there are some denatured soluble proteins which CCT will simply not bind (Melki and Cowan, Mol. Cell Biol. 14, 2895–2904, 1994). CCT, like GroEL, possesses ATPase activity and the ATPase domain on each CCT subunit is the region showing highest homology with GroEL. There is no GroES like co-chaperonin known for any of the type II chaperonins.
The significantly greater complexity of CCT over and above that of GroEL might suggest that CCT possesses affinity for a wider spectrum of unfolded substrates than GroEL. This does not appear to be the case and therefore an alternate view on the reason for the greater complexity of CCT is that it performs a more complex role within eukaryotic cells than GroEL does in prokaryotic cells. Phylogenetic analysis points to an early divergence of prokaryotic and eukaryotic Chaperonins (Kubota et al, Curr.Biol., 4, 89–99, 1994) and if CCT evolved at a similar time to the emergence of the cytoskeleton then a specialist actin/tubulin binding function may well have evolved for this Chaperonin family member (Willison and Kubota,The Biology of Heat Shock Proteins and Molecular Chaperones, CSH Press, N.Y., U.S.A 1994).
The vast majority of analysis on Chaperonin substrates has been performed on GroEL, and consequently an appreciation of the breadth of substrates of CCT is more limited. Whilst several known substrates of CCT and CCT analogues have been reported, namely actin, tubulin neurofilament, firefly luciferase, chromaffin membrane components and hepatitis B virus capsid several other legitimate substrates of CCT remain to be identified (Hynes et al, Electrophoresis 17, 1720–1727, 1996). Recent studies have shown that a protein SRB is homologous to CCTδ and may be responsible for binding and enhancing the interaction of TRP-185 with TAR-RNA in HIV infected cells (Wu-Baer et al, J. Biol. Chem. 271, 4201–4208, 1996).
Very little data generated to date has pointed towards the structure, assembly or existence of intermediate sized CCT complexes. There have been two reports which suggest that perhaps CCT subunits act independently of the main 16 subunit double toroid structure.
In Xenopus (Dunn and Mercola, 1996) have shown that two subunits (a and y) are developmentally regulated and that high levels of expression in the neural crest tissues might represent the site of novel substrates for CCT.
Further evidence of the existence of micro-complexes comes from analysis of CCT in ND7/23 cells undergoing differentiation to a neuronal phenotype. Roobol et al have shown that CCTa enters neuritic processes and co-localises with actin at the leading edge of growth cone structures whereas three other CCT subunits remain predominantly in a perikaryl cytoplasmic region of the cell (Roobol et al 1995).
CCT is significantly more complex than GroEL in terms of subunit specificity, developmental expression and cellular localisation and recently further evidence of control of activity has come to light with the discovery of a novel post translational modification namely tyrosine adenylylation of CCT. Further evidence of post translational modification has been reported following isoelectric focusing analysis of CCT complexes where evidence of subunit isoforms was evident. If CCT does perform more complex cellular functions than just folding it is reasonable to assume that CCT subunits might be phosphorylated, adenylylated, myrisytolated etc., giving rise to apparent isoforms on 2D gel analysis, a phenomena manifest in proteins which are control points in cellular metabolism.